Research

Our research effort is primarily focused on the study of
biological clocks, using zebrafish as a model system. Our early work was amongst
the first to show that the majority of tissues and cells of the body contain
independent clocks, which regulate the timing of fundamental aspects of cell
biology. An unusual feature of the fish circadian system is that most cells are
also directly light responsive. Some projects in the laboratory are aimed at
identifying the molecules involved in this unusual light response, while others
focus on the processes that the clock regulates. We employ a variety of
molecular, cellular and biochemical techniques for these cell-based studies,
including single cell luminescent imaging in the newly established Luminescent
Imaging Facility here at UCL. Additional research projects include studies in
the blind cavefish, Astyanax Mexicanus, where we are exploring both molecular
and behavioural rhythms in the laboratory here in London, as well as in the
caves of Northeastern Mexico.

HISTORY

ACCIDENTS AND GOOD LUCK. In
1996, Nick Foulkes
and myself at the IGBMC in Strasbourg, France decided to develop zebrafish as a
model system for a molecular examination of the circadian clock. Our starting
position was quite clear. Following on from the earlier work of Greg Cahill in Houston (who sadly passed away in
2008), we believed the circadian clock was localized to both the eyes and pineal
gland (Cahill, 1996). The first aim was to isolate a circadian clock gene, which
could then be used as a marker of clock function. This we eventually achieved by
cloning a zebrafish homolog of the then recently isolated mouse CLOCK
gene (King et al., 1997). Initial expression studies showed that the
zebrafish CLOCK transcript (now called CLOCK1) oscillated in
both retina and pineal. We then examined CLOCK1 expression in a number
of other tissues, mainly as a negative control for our eye and pineal data.
These experiments were prior to the description of clock gene expression in
mammalian tissues, and our expectation was for minimal or no expression of clock
genes in the periphery. To our surprise, the CLOCK1 transcript was not
only expressed, but also oscillated in zebrafish organs with the same timing as
in the eye and pineal. The development of a simple organ culture system then
allowed us to demonstrate that this oscillation continued in vitro, and
so conclude that organs within the fish contained an autonomous circadian
oscillator (Whitmore et al., 1998). The situation in fish is,
therefore, similar to that described in Drosophila (Plautz et
al., 1997).

The next obvious question was, how do these
peripheral clocks entrain to the environmental light-dark cycle? Though it
seemed somewhat insane, we went ahead and placed cultured organs (initially
hearts and kidneys) on a light-dark cycle in an incubator, illuminated by a
fibre optic. Matching organs from sibling fish on the same light-dark cycle were
dissected and placed into a neighbouring incubator, but on a reverse light-dark
cycle. The oscillation in CLOCK1 gene expression was then determined in
both groups by RNase protection assays and showed that this organ clock could be
re-entrained in vitro simply by changing the lighting regime.
Therefore, these tissues not only contain a clock, but also the photopigments
required to detect light, as well as the signal transduction machinery to set
that clock (Whitmore et al., 2000).

Thanks to the kind help of Uwe Straehle (then in
Strasbourg), we gained access to a number of embryonic zebrafish cell lines. The
first of these we examined (PAC2 cells made initially by Nancy Hopkins' lab at
MIT) showed a high level of CLOCK1 gene expression. When placed on a
light-dark cycle, these cells oscillated with a circadian period and timing
identical to that found in zebrafish organs. We now had access to a cell line
that contained a clock and the phototransduction machinery necessary to set that
clock (Whitmore et al., 2000).

RECENT STUDIES

Since these early studies, we
have generated a number of luminescent zebrafish cell lines, which we use
routinely to monitor clock function (Vallone et al., 2004). The
entrainment study described above can now be performed with relative ease by
maintaining these cells under different lighting conditions and monitoring
bioluminescence in an automated fashion, as shown below, in our
Per1-luciferase cells. These luminescent cell lines provide an
invaluable tool for our clock studies, and allow students and postdocs in the
laboratory to get plenty of sleep. Using very sensitive photon counting cameras
to measure luminescence, we have gone on to image gene expression rhythms in
single cells. These experiments have revealed an unexpected (at least, to us)
level of stochastic noise within these cellular clocks (Carr and Whitmore,
2005). Additional studies have elucidated the key role played by one of the
cryptochromes, Cry1a, in setting this clock to light (Tamai et
al., 2007). Our studies in zebrafish embryos have shown the incredibly
early stage at which the clock starts (Dekens and Whitmore, 2008), and even more
fascinating, how extremely light responsive a blastula stage fish embryo is,
only hours after fertilisation (Tamai et al., 2004). Our current
studies build on these early observations and are leading us into exciting new
directions, such as clock control of the cell cycle and, even more unexpectedly,
studies on clock function in the caves of Northeastern Mexico.